CN1006903B

CN1006903B - Production method for interleukin-2

Info

Publication number: CN1006903B
Application number: CN86101353A
Authority: CN
Inventors: 北野一昭; 藤本茂
Original assignee: Takeda Chemical Industries Ltd
Current assignee: Takeda Pharmaceutical Co Ltd
Priority date: 1985-03-11
Filing date: 1986-03-10
Publication date: 1990-02-21
Also published as: KR860007376A; JPS61205497A; EP0194818A2; ES8705041A1; CN86101353A; IL78074A0; ES552846A0; JPH0646957B2; EP0194818A3; DK104986A; KR930002737B1; DK104986D0; US4935356A

Abstract

The present invention provides an improvement in a method for producing interleukin-2 by cultivating a transformant of Escherichia coli capable of producing interleukin-2 into a medium, which comprises inoculating said Escherichia coli into the medium of pH from about 4.8 to 6.0 and growing it while maintaining this pH range. By the method of the present invention, the interleukin-2 productivity is considerably improved.

Description

Production method for interleukin-2

The relevant a kind of method of producing interleukin II of the present invention.

Interleukin II [be designated hereinafter simply as IL-2, be also referred to as T cell growth factor (TCGF)] is by stimulation T cells such as lectin, allogenic antigens and a kind of lymphokine that produces.(" science " magazine, 193 volumes, 1007 pages, 1976)

Utilize IL-2 can obtain many clones, such as: clone of T killer cell clone, t helper cell clone and natural killer cell or the like.(" nature " magazine, 268 volumes, 154 pages, 1977).Except it being directly used in the clone of T cell or natural killer cell, IL-2 also can be used for the in-vitro multiplication of the killer T cell of antigen-specific.The killer T cell of this antigen-specific can be discerned and destroy such as specific antigens such as tumour antigens.Can also be by being transferred to animal to the such tumour-specific killer T cell of propagation, suppress growth of tumor (see " IMMUNOLOGY KEY WORDS INDEX, 125 volumes, 1904 pages, 1980).

These test-results mean that using IL-2 has very big potentiality as a kind of anti-tumor agent.People are known, and IL-2 has the shortage of promotion thymus gland nude mice, the function that helper cell is recovered (see " European IMMUNOLOGY KEY WORDS INDEX, 10 volumes, 719 pages, 1980); Also have and promote killer T cell to induce the function of generation helper cell (seeing " nature " magazine, 284 volumes, 278 pages, 1980).Therefore, IL-2 is expected to be used to treat immunodeficiency diseases.

Can obtain people's IL-2 from people's T cell, but its quantity is very small.Yet, owing to the development of gene recombination technology in recent years, can contain the intestinal bacteria of IL-2 gene recombined vector by cultivation, obtain human IL-2 as biological activity protein.(seeing " nature " magazine, 302. volumes, 305 pages, nineteen eighty-three and " nucleic acids research ", 11 volumes, 4307 pages, nineteen eighty-three)

Usually produce human IL-2's method,, be not suitable for industrial application because its productive rate is low.In this case, the big quantity research of present inventor cultivate the colibacillary method can produce IL-2.Discovery is cultivated intestinal bacteria under the acidic conditions of PH4.8-6.0, with regard to can improve considerablely the productive rate of IL-2 but colibacillary fermentation generally be under pH is about the neutrallty condition of 6.5-7.5, to carry out, because think that intestinal bacteria tend under neutrality or slight alkalinity condition growth and (see " biochemical engineering ", the Tokyo University publishes, the 25-26 page or leaf, nineteen sixty-five).

Based on above-mentioned discovery, inventor further research has obtained achievement of the present invention.

The invention provides a kind of method of producing interleukin II.In substratum, cultivate the intestinal bacteria transformant that to produce interleukin II exactly, it is inoculated in the substratum of PH4.8-6.0, it is grown in this PH scope.

Above the said intestinal bacteria that can produce IL-2 have the IL-2 gene that obtains with gene recombination technology.

The present invention recommends to use mammiferous IL-2 gene, preferred people's IL-2 genes encoding, and its amino-acid sequence (contains 1-133 amino acid) as shown in Figure 1.Wherein the base sequence of this gene (password 1-133) is shown in Fig. 2.

Also be included as more following its physiologically active peptide coded DNA similar to IL-2: its amino-acid sequence DNA as shown in Figure 1, wherein halfcystine password (as: 125 halfcystines) is replaced by Serine or Threonine password; Another has the DNA of the amino-acid sequence identical with IL-2, but the fragment of four amino acid codings of its N end is removed.(seeing Japanese patent laid-open publication gazette 126088/1985).

Said gene (DNA) must have a promotor or a plurality of upstream promoter.The available promotor comprises tryptophane (trp) promotor, lactose (Lac) promotor, protein chain elongation factor Tu(tuf B) promotor and rec A promotor or the like.What the present invention preferably used is trp promoter.

Above mentioned gene and promotor generally are transformed in the plasmid as conversion carrier, and that the most frequently used is pBR 322.It is the redundant organism (seeing " gene ", 2 volumes, 95 pages, 1977) of plasmid Col El.But also available other plasmid is as long as their reproducible, reservations in Bacillus coli cells just can.For example: pBR 313(sees " gene ", 2 volumes, 75 pages, 1977); PBR 324 and pBR 325(see " gene ", 4 volumes, 121 pages, 1978); PBR327 and pBR328(see " gene ", 9 volumes, 287 pages, 1980); PKY 2289(sees " gene ", 3 volumes, 1 page, 1978); PKY 2700(sees " biological chemistry ", 52 volumes, 770 pages, 1980); PACYC 177 and pACYC 184(see " bacteriology magazine ", 134 volumes, 1141 pages, 1978); And pRK 248 pRK 646 and pDF 41(see " Enzymology method ", 68 volumes, 268 pages, 1979).

As having of expression vector: λ gt λ c phage, (" Proc. Natl. Acad. Sci.USA ", 71 volumes, 4579 pages, 1974) λ gt λ B phage, (the same of belonging to λ gt system, 72 volumes, 3416 pages, 1975) λ Dam phage (sees " gene ", the 1st volume, 255 pages, 1977); Block imperial carrier (" science ", 196 volumes, 161 pages, 1977 and " Journal of Virology ", 29 volumes, 555 pages, 1979) and filobactivirus.

Above-mentioned expression vector can be set up (seeing " nature ", 302 volumes, 305 pages, nineteen eighty-three and " nucleic acids research ", 11 volumes, 4307 pages, nineteen eighty-three) with conventional method.

, transform as host bacteria with intestinal bacteria with being connected with human IL-2's expression carrier.The redundant organism of e. coli k-12 bacterial strain from operation and security standpoint, is good especially.For example: coli strain PR13, C-4,294, DH1, W 3110 and C600 use the result fine.

Intestinal bacteria C-4 be one from the isolated bacterial strain of PR13 (seeing " bacteriology magazine ", 97 volumes, 1522 pages, 1969), it is the maternal bacterial strain of K-12() redundant organism.It has following bacterial characteristics:

A) microscopic features

1) shape and size: as normal e. coli k-12 bacterial strain, be single pole bacterium or diplobacterium, 0.5-1.0 * 2-5 μ m does not show polymorphism.

2) mobility: movable.

3) flagellum: tool peritrichous.

4) gramstaining: feminine gender.

5) sporulation: do not have.

6) acid resistance: not acidproof

B) growth conditions in different substratum

1) the bouillon agar plate is cultivated: bacterium colony is little, and is flat, and ring-type is translucent and glossy.

2) bouillon agar slant culture: bacterium colony median size, flat, translucent and glossy slightly.

3) meat soup liquid culture: it is medium to grow, and generates the homogeneous suspension.

4) bouillon gelatine stab culture: grow into the disperse state of homogeneous, do not cause gelatine liquefication.

5) litmus milk: do not condensed or peptonize, pH value also changes.

C) physiology characteristic

1) nitrate reduction: the positive

2) denitrification: feminine gender

3) MR test: the positive

4) VP test: feminine gender

5) indoles produces: the positive

6) hydrogen sulfide produces: feminine gender

7) starch hydrolysis: feminine gender

8) citric acid utilization: feminine gender

9) inorganic nitrogen-sourced utilization:

ⅰ) SODIUMNITRATE: the positive

ⅱ) sulfuric acid amine: the positive

10) adding lustre to property: and do not find to have water colo(u)r to produce

11) urease: feminine gender

12) oxydase: feminine gender

13) catalase: feminine gender

14) growth conditions:

ⅰ）PH：4.5-10.0

ⅱ) temperature: 18-47 ℃

15) behavior in oxygen: obligate anaerobic

16) O-F test: the positive

17) utilize different carbohydrates to produce acid and product oxygen ability: to see Table 1

Table 1

The sugar acid producing ability produces the oxygen ability

L-arabinose++

The D-wood sugar--

D-glucose++

The D-seminose++

D-fructose++

The D-semi-lactosi--

Maltose--

Sucrose--

Lactose++

Trehalose++

The D-sorbyl alcohol++

D-N.F,USP MANNITOL--

Inositol--

Glycerine++

Starch--

This bacterial strain (intestinal bacteria C-4) is stored in the fermentation research institute (FRI) of Ministry of International Trade and Industry industrial technology administration on February 16th, 1985, and storing number is FERMP-8101; The preservation strain that this preservation thing has been changed into the energy budapest treaty promptly is kept at FRI with FERMBP-966; And be deposited in Osaka fermentation research institute (IFO) with IFO-14421, and be stored in the CGMCC of China, storing number is CGMCC0085.Store number on January 28th, 86.

Intestinal bacteria 294 are known bacterial strain (" Proc. Natl. Acad. Sci.USA ", 73 volumes, 4474 pages, 1976), have been stored in IFO, and storing number is IFO-14171.

W3110 and C600 also are known bacterial strains; They are at the U.S. ATCC bacterial strain catalogue first roll of nineteen eighty-two the 15th edition, store number to be respectively ATCC27325 and ATCC27324.

Nineteen sixty-eight " nature " magazine, 217 the volume, 1110 pages, narrated the DH1 bacterial strain.

Transform the host e. coli cell with expression plasmid, when production can be made the Bacillus coli cells of IL-2, available " molecular biology magazine ", 53 volumes, 159 pages, 1970; " Enzymology method ", 68 volumes, 253 pages, 1979; And " gene ", 3 volumes, 279 pages, the method for being narrated in 1978 transforms.

The intestinal bacteria that can produce IL-2 are inoculated on the substratum of PH4.8-6.0, again culturing bacterium in this PH scope.The PH scope is 5.0 to 5.8 preferably, and growth is best during PH5.5.

Yet after bacterium fully grew, the PH in the substratum can be offset out above-mentioned scope and become sourer situation.

Forward and backward in medium preparation and sterilization, available mineral acid or alkali are regulated pH value.During the intestinal bacteria incubation growth, regulate PH all the time and make it remain on a specific scope.Because PH often will reduce in the microbial culture process, therefore, must add and regulate PH such as mineral alkalis such as ammoniacal liquor, sodium hydroxide and yellow soda ash, also can add mineral acids such as sulfuric acid if desired, in these materials, ammoniacal liquor is preferentially selected for use, because it also is the source of nitrogen in the substratum.

Through substratum commonly used is M-9 substratum and the M-03 substratum that has replenished glucose and casamino acids.(nutrient media components sees Table 2).Yet, not only available these two kinds of substratum, any substratum as long as bacterium can produce IL-2 thereon, all can be used.To being connected with the recombinant chou such as promotors such as trp promoters, can add such as 3-β-reagent such as indyl vinylformic acid increases the efficient of promotor.During the microbial culture, if desired, also can add materials such as glucose and casamino acids.Bacillus coli cells for preferential breeding reorganization can use them that it is had the medicament (as: tsiklomitsin etc.) of resistance according to the type of contained gene in their plasmids.

Table 2 can be used the example of substratum

Composition improvement M-9 substratum M-03 substratum

Glucose 10 grams per liters 10 grams per liters

NaHPO 6 grams per liters-

KHPO 3 grams per liters 3 grams per liters

NaCl 0.5 grams per liter 0.5 grams per liter

NHCl 1 grams per liter 1 grams per liter

MgSO7HO 0.34 grams per liter 0.34 grams per liter

Casamino acids 10 grams per liters 10 grams per liters

Culture temperature is generally 15 to 45 ℃.Alternating temperature can obtain higher productive rate in the following manner: keep about 37 ℃ up to growth mid-term, reduce temperature to 20～30 ℃ again.

Usually make the culture ventilation with stirring.In the substratum to keep oxygen to contain 5%(v/v approximately), or higher saturated oxygen concentration is advisable, because this will increase the output of IL-2.If during microbial culture, use pure oxygen in conjunction with ventilation, may be more effective.

Aforesaid method is produced the amount of IL-2, and the clone of available dependence IL-2 is measured.Well-known, the human IL-2 promotes rat and mouse to rely on the propagation of IL-2 cell, also promotes the people to rely on the propagation of IL-2 cell (seeing " Immunological Review ", 51 volumes, 257 pages, 1980).Therefore, can personnel selection, the clone of the dependence IL-2 of rat or mouse (" IMMUNOLOGY KEY WORDS INDEX, 130 volumes, 981-988 page or leaf, nineteen eighty-three).

Through quite long period, the clone that mouse can be relied on IL-2 is made stable especially subculture thing, can obtain having the highly data of repeatability from them.

In this manual, measure the amount that produces IL-2 with the mouse cell that relies on IL-2.According to this method, with what of the mouse cell picked-up radio isotope thymidine that relies on IL-2, as a kind of indication (" biochemical biophysical research communication ", 109 volumes, 363 pages, nineteen eighty-two) of quantity.

According to different condition, the present invention can extract the IL-2 that culturing cell produces with different methods.For example: the 1) bacterial cell of collect cultivating by ordinary method, it is suspended in the damping fluid of a kind of protein denaturant that contains Guanidinium hydrochloride, under the ice-cold condition, stir suspension, it is centrifugal, obtain containing the supernatant liquor of IL-2.2) culturing cell with collection is suspended in the damping fluid, makes cell rupture with ultrasonic wave, and is after N,O-Diacetylmuramidase and/or freeze thawing treatment that the gained suspension is centrifugal, obtains containing the supernatant liquor of IL-2.

Can be used in combination conventional separation and separate IL-2 and make it purifying from above-mentioned supernatant liquor with method of purification, used the whole bag of tricks is as follows: based on deliquescent method, as: saltout and organic solvent deposit; Mainly based on the molecular weight diverse ways, as; Dialysis, ultrafiltration, gel-filtration and SDS-polyacrylamide gel electrophoresis; Based on electrically charged diverse ways, as: ion exchange chromatography; Based on pathoklisis, for example: affinity chromatography; Based on hydrophobic difference, as: the anti-phase high pressure liquid chromatography; Based on the iso-electric point diverse ways, as: the iso-electric point electrophoresis.Because human IL-2's albumen is highly hydrophobic, the hydrophobicity chromatogram is particularly used the flyback type post, is obvious and effective when this albumen of purifying.

The IL-2 albumen of above-mentioned purifying can allow to discern and the killer T cell of the antigenic antigen-specific of destroyed tumor in external preferential propagation; The IL-2 albumen of above-mentioned purifying also can make those natural killer cells that can destroy no antigen sensibility tumor at in-vitro multiplication.In addition, when these killer T cells were transferred in vivo, the human IL-2 that the present invention produces just unavoidably was inoculated in the body simultaneously, thereby had improved the anti-tumor capacity of these cells.Based on this reason, above-mentioned IL-2 albumen can be used for preventing canceration, treatment tumour and immunodeficiency diseases.It can be used for such as homoiothermy animals such as mouse, rat, rabbit, dog, cat, pig, horse, sheep, ox and people.

When above-mentioned IL-2 albumen is made the control tumour medicine, can be with known drug carrier dilution back oral or inject outward with injection form enteron aisle, or make capsule.In addition, it can use separately or with as previously mentioned propagation kill and wound the R cell or natural killer cell is used in combination.

In addition, when using in the above described manner, above-mentioned IL-2 albumen is similar especially to known natural human IL-2's biological activity; Because its dissociation constant from the IL-2 acceptor is very little, therefore, even very low consumption, effect also is gratifying.

Fig. 1 represents human IL-2's amino-acid sequence (X represents methionine(Met) or hydrogen).

Fig. 2 represents an example of human IL-2's gene DNA order.

To the present invention be described in detail in detail with preferred embodiment and reference example below.

Fermentation research institute (FRI) and Osaka fermentation research institute (IFO) of the registered industrial technology administration at Ministry of International Trade and Industry of the representative transformant described in the following example, storage number is shown in following table.

Store the FRI IFO CGMCC of mechanism

Transformant (storing the date)

Intestinal bacteria FERM IFO-14422 CGMCC0086

C-4/PTF4 BP-967

(on February 16th, 1958) (on January 28th, 1976)

The known bacterial strain of intestinal bacteria FERM IFO-14299

DH1/PTF4 BP-628

(on April 6th, 1984)

Example 1

The expression plasmid pTF4 that contains human IL-2's building stone is from intestinal bacteria DH1/pTF4(FERM BP-628) separate (seeing disclosed European patent № .145390), be to use Birnboim, people's such as H.C. method (is seen " nucleic acids research ", 7 volumes, 1513 pages, 1979).According to Cohen, people's such as S.N. method is seen " Proc. Natl. Acad. Sci.USA " with described plasmid transformation escherichia coli PR13(, 69 volumes, 2110 pages, 1972 years).In one 200 milliliters Erlenmeyer flask, add 50 milliliters (PH7.0) and contain 1%Bacto-trypfon(Difco Laboratories USA), 0.5% bacterium-yeast extract paste (Difco), the substratum of 0.5% sodium-chlor and 5 mg/litre tetracycline hydrochloride, the transformant of gained is inoculated on the substratum, then with it 37 ℃ of following overnight incubation.Adorn in 200 milliliters of Erlenmeyer flasks of 30 milliliters of substratum in again each nutrient solution being inoculated into respectively, substratum is the improvement M-9 substratum that contains 1 mg/litre hydrochloric acid vitamins B, it was cultivated 4 hours at 37 ℃, cultivated 4 hours at 30 ℃ again, cultivated 10 hours at 25 ℃ at last, selecting obviously high bacterial strain of IL-2 productive rate, promptly is intestinal bacteria C-4/pTF4.

Again the intestinal bacteria C-4/pTF4 cell inoculation to of gained is equipped with in 250 milliliters of Erlenmeyer flasks of 50 milliliters of (PH7.0) substratum, include the 1%Bacto-trypton(Difco product), 0.5% bacterium-yeast extract paste (Difco product), 0.5% sodium-chlor, 5 mg/litre tetracycline hydrochloride, 37 ℃ of overnight incubation, obtain initial incubation liquid.In the fermentor tank of 85 liter capacities, inject 2.5 liters of substratum that contain the M-03 of 1 mg/litre vitamins B respectively; After the sterilization, the PH of the substratum of 8 jars is transferred to 7.5,7.0,6.5,6.0 respectively, 5.5,5.0,4.8 and 4.5 with ammoniacal liquor or 5N sulfuric acid.Be inoculated in each fermentor tank with 125 milliliters of initial incubation liquid, in 37 ℃, cultivate,, make each fermentor tank keep specific pH value with ammoniacal liquor or continuous adjustment of 5N sulfuric acid with the rotating speed stirring of 2.5 liters of/minute ventilations and per minute 1300.When glucose content reduces to 0.5%, add 1% glucose and 1% casamino acids between incubation period, continue to cultivate; The result is shown in table 3.PH is at the pH value that usually adopts at PH7.0(of the IL-2 productivity ratio between the 4.6-6.0) time increase 2-5 doubly.

PH was to the influence of IL-2 productive rate when table 3 was cultivated

(37 ℃ of cultivations)

PH IL-2 productive rate *

4.5 15

4.8 240

5.0 400

5.5 520

6.0 330

6.5 150

7.0 100

7.5 50

* productive rate is represented is to be 100 o'clock ratio with the PH7.0 productive rate.

Example 2

Prepare the substratum of 8 different PH with the mode identical with example 1, the initial incubation liquid that example 1 is obtained is inoculated on this substratum, then in ventilation with shake under the condition, cultivates 24 hours with alternating temperature.Promptly cultivate down at 37 ℃ earlier; When culture reaches 500Klett unit, be cooled to 30 ℃; When reaching 1000Klett unit, reduce temperature to 25 ℃, till the cultured continuously to 24 hour.The gained result is shown in table 4.The IL-2 productivity ratio is at PH7.0 between 4.8-6.0 for PH, and the IL-2 gain in yield 3-7.5 during 37 ℃ of following constant temperature culture doubly.

PH was to the influence of IL-2 productive rate when table 4 was cultivated

(alternating temperature cultivation)

PH IL-2 productive rate *

4.5 10

4.8 300

5.0 530

5.5 750

6.0 510

6.5 180

7.0 130

7.5 60

That * productive rate is represented is PH7.0, and 37 ℃ of following productive rates are 100 o'clock ratio.

Example 3

With the pTF4 that is integrated with human IL-2's structure gene as expression plasmid, according to the method for example 1, transformed into escherichia coli bacterial strain 294, DH1, W3110 and C600.The transformant that obtains is inoculated in the initial medium identical with example 1,37 ℃ of overnight incubation.Respectively 2.5 liters of M-9 substratum that contain 1 mg/litre hydrochloric acid vitamins B are injected 8 volumes and be 5 liters fermentor tank, the PH of substratum in 4 fermentor tanks is transferred to 5.5, remain 4 and transfer to 6.5.All inoculate for every jar, in example with 125 milliliters initial conversion body nutrient solution

2 similarity condition is cultivated down, and the result is shown in table 5.

Table 5 PH is to the influence of IL-2 productive rate in the different transformant.

Transformant (intestinal bacteria) PH6.5 PH5.5

294/PTF4 100 200

DH1/PTF4 100 260

W3110/PTF4 100 310

C600/PTF4 100 280

In all transformant, the IL-2 productive rate during PH5.5 increases more than 2 times during all than PH6.5.

Example 4

As the substratum of 8 different PH of example 1 preparation, under the temperature condition identical,, keep specific pH value invariable with the 5N NaOH aqueous solution or 5N sulfuric acid between incubation period with every kind of substratum cultured continuously 24 hours with example 2.The result is shown in table 6.Increase 5-7 during the productivity ratio PH7.0 of IL-2 doubly during PH4.8-6.0.

Table 6 PH is to the influence of IL-2 productive rate

(PH regulates with NaOH, keeps constant)

PH IL-2 productive rate *

4.5 20

4.8 275

5.0 560

5.5 725

6.0 530

6.5 210

7.0 100

7.5 35

* productive rate is to be 100 o'clock ratio with PH7.0.

Example 5

The every kind of nutrient solution (PH5.5 or 7.0) that obtains in the example 2 is centrifugal, collect bacterial cell, freezing at-80 ℃ then.Get every kind of frozen cell 12 gram, it be suspended in 100 milliliters equably include 7M Guanidinium hydrochloride and 0.1M Tris-HCl(PH7.0) extraction liquid in, shook 1 hour at 4 ℃.Every kind of molten cytosol that will obtain then centrifugal 20 minutes with 28000xg obtains supernatant liquor.Each supernatant liquor with 0.01M Tris-HCl damping fluid (PH8.5) dialysis, with 19000xg centrifugal 10 minutes then, is obtained in the dialysis

Clear liquid.It is crossed one with 0.01M Tris-HCl damping fluid (PH8.5) equilibrated DE52 post (the DEAE Mierocrystalline cellulose is a Britain Whatman company product), the post material is with protein adsorption again; Use a LINEAR N aCl concentration gradient (0-0.15M NaCl, 1 liter) wash-out IL-2 then.With a YM-5 film (from Amicon Co., USA) each active part is concentrated to about 5 milliliters, use 0.1M Tris-HCl(PH8.0 at one then)-1M NaCl damping fluid equilibrated Sephacryl S-200(Sweden, Pharmacia company product) on the post (500 milliliters of volumes), gel-filtration.Adsorb above-mentioned concentrated solution with a ultramicropore RPSC post (Altex Co., USA product), again it is advanced the high pressure liquid chromatography post, with trifluoroacetic acid-acetonitrile system wash-out.

Keep following condition:

Post: ultramicropore RPSC(4.6 * 75mm)

Column temperature: 30 ℃

Elutriant A:0.1% trifluoroacetic acid-99.9% water

Elutriant B:0.1% trifluoroacetic acid-99.9% acetonitrile

Used 68%A+32%B in elution program: 0-25 minute

Used 55%A+45%B in 25-35 minute

Used 45%A+55%B in 35-45 minute

Used 30%A+70%B in 45-48 minute

Use 100%B after 48 minutes

Elution rate: 0.8 ml/min

Observation wavelength: 230 nanometers

The about active part that obtained in 39 minutes of wash-out is collected, and lyophilize obtains human IL-2's albumen of white powder.

Obtain 5.2 milligrams in IL-2 albumen from the bacterium of cultivating at PH7.0, and when PH5.5 cultivates, obtain 12.7 milligrams.Two kinds of situation purity of protein are 99%(and measure with densometer).The albumen that obtains under two kinds of situations, chemical property does not have difference.

Reference example

The intestinal bacteria C-4/pTF4 that use-case 1 obtains removes plasmid according to people's such as Bouanchaud method with ethidium bromide, obtains intestinal bacteria C-4 bacterial strain (" general microbiology magazine ", 54 volumes, 417 pages, nineteen sixty-eight).

Claims

1, in substratum, cultivates the method for the intestinal bacteria transformant production interleukin II that can make interleukin II, it is characterized in that this transformant that contains the e. coli k-12 bacterial strain of dna upstream promotor is inoculated in the substratum of pH4.8-6.0, under aeration condition, remain in this pH scope and make bacterial growth.

2, method according to claim 1, intestinal bacteria transformant wherein contain the coding DNA of the interleukin II of promising following amino-acid sequence:

1

X-Ala?Pro?Thr?Ser?Ser?Ser?Thr?Lys?Lys?Thr?Gln?Leu?Gln

20

Leu?Glu?His?Leu?Leu?Leu?Asp?Leu?Gln?Met?Ile?Leu?Asn

Gly?Ile?Asn?Asn?Tyr?Lys?Asn?Pro?Lys?Leu?Thr?Arg?Met

40

Leu?Thr?Phe?Lys?Phe?Tyr?Met?Pro?Lys?Lys?Ala?Thr?Glu

60

Leu?Lys?His?Leu?Gln?Cys?Leu?Glu?Glu?Glu?Leu?Lys?Pro

Leu?Glu?Glu?Val?Leu?Asn?Leu?Ala?Gln?Ser?Lys?Asn?Phe

80

His?Leu?Arg?Pro?Arg?Asp?Leu?Ile?Ser?Asn?Ile?Asn?Val

100

Ile?Val?Leu?Glu?Leu?Lys?Gly?Ser?Glu?Thr?Thr?Phe?Met

Cys?Glu?Tyr?Ala?Asp?Glu?Thr?Ala?Thr?Ile?Val?Glu?Phe

120

Leu?Asn?Arg?Trp?Ile?Thr?Phe?Cys?Gln?Ser?Ile?Ile?Ser

133

Thr?Leu?Thr

3, be a trp promoter or a plurality of trp promoter according to the promotor that the process of claim 1 wherein.

4, be 5.0 to 5.8 according to the pH value that the process of claim 1 wherein.

5, according to the process of claim 1 wherein that the pH value of substratum regulates as alkaline matter with mineral alkali.

6, according to the method for claim 5, mineral alkali wherein is an ammoniacal liquor.

7, be the M-9 substratum (i.e. Gai Liang M-9 substratum) that adds glucose and casamino acids according to the substratum that the process of claim 1 wherein.

8, begin under about 37 ℃, to carry out according to the process of claim 1 wherein to cultivate, cool the temperature to 20 to 30 ℃ mid-term to growth.